1. Field of the Invention
The invention relates to a process for obtaining microorganisms containing peptide amidase, microorganisms obtained therewith, peptide amidases contained in them and the use thereof.
The invention relates in particular to a screening process for microorganisms exhibiting peptide amidase activity in accordance with the generic part of claim 1; microorganisms obtained according to this process and deposited in conformity with claims 2-4; peptide amidases which can be isolated from the microorganisms according to claims 5-7 and the use thereof.
2. Background information
The following publications are cited regarding the state of the art:
(1) DE-OS 36 29 242, PA1 (2) K. Breddam, Carlsberg Res. Commun. 49 (1984) 535-554, PA1 (3) DE patent 40 14 564 and PA1 (4) Y. Nishida et al., Enzyme Microb. Technol., 6 (1984), 85-90. PA1 Splitting off of the C-terminal amino group from peptide amides and N-terminally protected amino acid amides; PA1 No splitting of peptide bonds; PA1 Optimum pH at 7.5.+-.1.5; PA1 Good stability in the pH range between pH 6.0 and pH 9.0; PA1 The optimum temperature is 30.degree. C. at a pH of 7.5; PA1 Slight inhibition by inhibitors from serine proteases, especially phenylmethane sulfonyl fluoride; PA1 The molecular weight is 23,000.+-.3,000; PA1 Aggregate formation is occasionally observed; PA1 The isoelectric point is approximately pH 9.5; PA1 The enzyme does not accept any D-amino acid groups in C-terminal position and the rate of hydrolysis thereby is distinctly less than in the case of L-amino acid groups. PA1 Splitting off of the C-terminal amino group from peptide amides and N-terminally protected amino acid amides; PA1 No splitting of peptide bonds; PA1 Optimum pH at 6.0.+-.0.5; PA1 Good stability in the PH range between pH 7 and pH 8; PA1 The optimum temperature is 35-40.degree. C. at a pH of 7.5; PA1 Inhibition of serine groups by inhibitors such as phenylmethane sulfonylfluoride as well as in particular 4-(2-aminoethylbenzylsulfonylfluoride) (Pefabloc); PA1 The molecular weight is approximately 38,000 daltons (determined by gel filtration); PA1 The isoelectric point is approximately pH 5.8. PA1 Purification of &gt;500 at a yield of &gt;60% PA1 A molecular weight of 38000 da (gel filtration) PA1 The isoelectric point is approximately pH 5.8 PA1 Temperature optimum between 37-45.degree. C. PA1 pH optimum between 5-6.5 PA1 Temperature-stable at 20, 30 and 37.degree. C. over 3 days PA1 At pH 7-8 the enzyme is stable at 30.degree. C. over 7 days PA1 Upon the addition of 20% DMF the peptide amidase exhibits after 24 h a residual activity of 32% at a pH of 7.5 PA1 Serine group decisive for enzyme activity.
A peptide amidase is an enzyme which catalyzes the selective hydrolysis of a C-terminal amide function in a peptide amidase, that is, accelerates the following conversion: ##STR1##
Here, R' signifies a protective group for n=0 and for n&gt;0 any amino acid, a protective group or H; n stands for zero or any whole number, R.sub.x are the side chains of the amino acids for n&gt;0 whereas R.sub.1 signifies the side chain of the C-terminal amino acid.
The selective splitting off of the C-terminal amino group of peptide amides is generally difficult to achieve by a chemical conversion since the peptide bond is also subject to a hydrolytic attack. This results in mixtures which are difficult to separate and in low yields.
Reference (1) teaches amidases for an enzymatic splitting off of the acid amide group which, on account of their a-amino acid amidase activity, can only be used, however, for the production of L-amino acids from a-unprotected D,L-amino acid amides. Peptide amides are not accepted.
Reference (4) teaches the continuous production of N-Ac-L-Met from N-Ac-D,L-methionine amide in an enzymatic process using Erwinia carotovera.
Erwinia carotovera does contain an amidase activity; however, it is limited exclusively to amides of methionine. Thus, the enzyme from Erwinia carotovera only "splits off amino acid amide" and is not a peptide amidase. Furthermore, the enzyme from Erwinia carotovera can obviously only convert N-acetylated amino acid amides, in which conversion it is a disadvantage that the Ac protective group can only be split off with difficulty or not at all.
On the other hand, peptidases are known which catalyze the hydrolytic splitting of the peptide bonds and of which it is only known that they have a certain secondary activity for splitting off the C-terminal amide protective group. An example of this is the carboxy peptidase Y, especially in chemically modified form (see reference (2)).
Thus, all these processes have serious disadvantages.
The state of the art according to reference(3) is also a peptide amidase which can be isolated from the flavedo of citrus fruits, especially of oranges. The peptide amidase described does not attack the peptide bond and catalyzes the splitting off of the free amino group from peptide amides. The peptide amidase known from (3) is characterized by the following parameters:
However, the isolated enzyme can be obtained from flavedo only in slight amounts and as a function of the season. More extensive studies also did not succeed, in spite of an approximately 500-fold enrichment, in preparing the protein in homogeneous form, so that molecular and genetic studies for improving the enzyme production were not able to be included due to lack of data.
However, this also renders the suggestion given in reference (3) moot--that a microbial production of the enzyme can be achieved in a known manner by gene technology manipulation. The problems in the presentation of the homogeneous form do not allow manipulations of gene technology.